Invariant Ser211 is involved in the catalysis of PD-L4, type I RIP from Phytolacca dioica leaves

Angela Chambery, Marianna Pisante, Antimo Di Maro, Erika Di Zazzo, Menotti Ruvo, Susan Costantini, Giovanni Colonna, Augusto Parente

Research output: Contribution to journalArticle

Abstract

Multiple sequence alignment analysis of ribosome inactivating proteins (RIPs) has revealed the occurrence of an invariant seryl residue in proximity of the catalytic tryptophan. The involvement of this seryl residue in the catalytic mechanism of RIPs was investigated by site-directed mutagenesis in PD-L4, type 1 RIP isolated from Phytolacca dioica leaves. We show that the replacement of Ser211 with Ala apparently does not influence the N-β-glycosidase activity on ribosomes (determined as IC50 in a cell-free system), but it reduces the adenine polynucleotide glycosylase activity (APG), assayed spectrophotometrically on other substrates such as DNA, rRNA, and poly(A). The ability of PDL4 to deadenylate polynucleotides appears more sensitive to the Ser211Ala replacement when poly(A) is used as substrate, as only 33% activity is retained by the mutant, while with more complex and heterogeneous substrates such as DNA and rRNA, its APG activity is 73% and 66%, respectively. While the mutated protein shows a conserved secondary structure by CD, it also exhibits a remarkably enhanced tryptophan fluorescence. This indicates that, although the overall protein tridimensional structure is maintained, removal of the hydrosyl group locally affects the environment of a Trp residue. Modelling and docking analyses confirm the interaction between Ser211 and Trp207, which is located within the active site, thus affecting RIP adenine polynucleotide glycosylase activity. Data accumulated so far confirm the potential involvement of Ser211 in the catalytic mechanism of type 1 RIP PD-L4 and a possible role in stabilizing the conformation of Trp207 side chain, which participates actively in the protein enzymatic activity.

Original languageEnglish
Pages (from-to)209-218
Number of pages10
JournalProteins: Structure, Function and Genetics
Volume67
Issue number1
DOIs
Publication statusPublished - Apr 2007

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Ribosome Inactivating Proteins
Type 1 Ribosome Inactivating Proteins
Catalysis
Tryptophan
Phytolacca
Substrates
Polynucleotides
Mutagenesis
Proteins
Poly A
Cell-Free System
Sequence Alignment
Glycoside Hydrolases
DNA
Site-Directed Mutagenesis
Ribosomes
Inhibitory Concentration 50
Sequence Analysis
Conformations
Catalytic Domain

Keywords

  • 3D modelling
  • Adenine polynucleotide glycosylases
  • Circular dichroic spectroscopy
  • Docking analysis
  • Fluorescence analyses
  • Ribosome inactivating proteins

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology

Cite this

Chambery, A., Pisante, M., Di Maro, A., Di Zazzo, E., Ruvo, M., Costantini, S., ... Parente, A. (2007). Invariant Ser211 is involved in the catalysis of PD-L4, type I RIP from Phytolacca dioica leaves. Proteins: Structure, Function and Genetics, 67(1), 209-218. https://doi.org/10.1002/prot.21271

Invariant Ser211 is involved in the catalysis of PD-L4, type I RIP from Phytolacca dioica leaves. / Chambery, Angela; Pisante, Marianna; Di Maro, Antimo; Di Zazzo, Erika; Ruvo, Menotti; Costantini, Susan; Colonna, Giovanni; Parente, Augusto.

In: Proteins: Structure, Function and Genetics, Vol. 67, No. 1, 04.2007, p. 209-218.

Research output: Contribution to journalArticle

Chambery, A, Pisante, M, Di Maro, A, Di Zazzo, E, Ruvo, M, Costantini, S, Colonna, G & Parente, A 2007, 'Invariant Ser211 is involved in the catalysis of PD-L4, type I RIP from Phytolacca dioica leaves', Proteins: Structure, Function and Genetics, vol. 67, no. 1, pp. 209-218. https://doi.org/10.1002/prot.21271
Chambery, Angela ; Pisante, Marianna ; Di Maro, Antimo ; Di Zazzo, Erika ; Ruvo, Menotti ; Costantini, Susan ; Colonna, Giovanni ; Parente, Augusto. / Invariant Ser211 is involved in the catalysis of PD-L4, type I RIP from Phytolacca dioica leaves. In: Proteins: Structure, Function and Genetics. 2007 ; Vol. 67, No. 1. pp. 209-218.
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abstract = "Multiple sequence alignment analysis of ribosome inactivating proteins (RIPs) has revealed the occurrence of an invariant seryl residue in proximity of the catalytic tryptophan. The involvement of this seryl residue in the catalytic mechanism of RIPs was investigated by site-directed mutagenesis in PD-L4, type 1 RIP isolated from Phytolacca dioica leaves. We show that the replacement of Ser211 with Ala apparently does not influence the N-β-glycosidase activity on ribosomes (determined as IC50 in a cell-free system), but it reduces the adenine polynucleotide glycosylase activity (APG), assayed spectrophotometrically on other substrates such as DNA, rRNA, and poly(A). The ability of PDL4 to deadenylate polynucleotides appears more sensitive to the Ser211Ala replacement when poly(A) is used as substrate, as only 33{\%} activity is retained by the mutant, while with more complex and heterogeneous substrates such as DNA and rRNA, its APG activity is 73{\%} and 66{\%}, respectively. While the mutated protein shows a conserved secondary structure by CD, it also exhibits a remarkably enhanced tryptophan fluorescence. This indicates that, although the overall protein tridimensional structure is maintained, removal of the hydrosyl group locally affects the environment of a Trp residue. Modelling and docking analyses confirm the interaction between Ser211 and Trp207, which is located within the active site, thus affecting RIP adenine polynucleotide glycosylase activity. Data accumulated so far confirm the potential involvement of Ser211 in the catalytic mechanism of type 1 RIP PD-L4 and a possible role in stabilizing the conformation of Trp207 side chain, which participates actively in the protein enzymatic activity.",
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